JP2009236848A - Nonspecific adsorption constraint material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nonspecific adsorption constraint material capable of detecting an objective molecular species sensitively while inhibiting the nonspecific adsorption of biomolecule having various molecular weight from high molecular weight to low molecular weight as much as possible. <P>SOLUTION: The nonspecific adsorption constraint material is formed by monomolecular film of compound expressed with general formula (1) on a surface of a substrate. HO(CH<SB>2</SB>CH<SB>2</SB>O)<SB>m</SB>-(CH<SB>2</SB>)<SB>n</SB>-SH...(1) Where, m represents an integer of 1-3, n represents an integer of 2-8. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention completely suppresses nonspecific adsorption of in vivo tissue components, cells, proteins, lipids, etc. on a substrate, and efficiently detects biomolecules such as target proteins, peptides, nucleic acids, etc. It is related with the nonspecific adsorption | suction suppression material for.

Response detection systems that immobilize biomolecules such as antibodies, antigens, and enzymes on the surface of a substrate and use the functions and specific reactions of these biomolecules are actively used for immunodiagnosis and specific biomolecule detection methods. I came. The development of DNA chips, protein chips, etc., that directly fix DNA to a substrate and detect target DNA has also been actively conducted.
In order to detect only biomolecules, proteins, and nucleic acids to be measured with high sensitivity from samples containing a large amount of contaminants such as biological tissues, cells, proteins, lipids, etc. It is necessary to devise ways to reduce the noise response due to non-specific adsorption of objects as much as possible.

As a method for preventing nonspecific adsorption of biomolecules into a substrate or a flow path, there is a method using a cyclodextran layer into which a carboxyl group is introduced (see, for example, Non-Patent Document 1), which is actually used ( CM5 chip and others, Biacore system (GE Healthcare Biosciences)).
However, the dextran layer is thick (approx. 100 nm), and the detection substance gets into the layer, causing problems with thermodynamic and kinetic parameters, or it can be seen that it is actually adsorbed. It may not be suitable for accurate measurement.
L. Stigh et al., “Methods for site controlled coupling to carboxymethyldextran surfaces in surface plasmon resonance sensors”, Biosensors & Bioelectronics 10 (1995) 813-822.)

On the other hand, methods such as applying a material having a polyethylene glycol (PEG) group to a substrate have been taken. It is known that the PEG group is highly hydrophilic and has an effect of preventing nonspecific adsorption. The PEG-containing polymer material is intended to suppress nonspecific adsorption by applying a polymer material on the surface of the substrate because the hydrophilic portion exhibits an effect of preventing nonspecific adsorption of proteins and the like. (See Patent Document 1 and Non-Patent Document 2)
JP 7-316285 A KL Prime and GM Whitesides, “Adsorption of proteins onto surfaces containing end-attached oligo (ethylene oxide): A model system using self-assembled monolayers, J. Am. Chem. Soc., 115 (1993) 10714-10721

In addition, even when aiming at suppression of nonspecific adsorption using a polymer having a PEG group, the nonspecific adsorption may not be completely suppressed because the polymer is bulky. An example has also been proposed in which a problem is solved by using two types of high molecular weight PEG, a molecular species having a higher molecular weight and a smaller molecule. (Patent Document 2)
JP 2006-177914 A

However, the technique described in Patent Document 1 uses polyoxyalkylene derivatives having different functional groups at both ends, and it is difficult to synthesize such derivatives. On the other hand, in the technique described in Patent Document 2, a PEG having a functional group that can be immobilized on the surface is first supported on the surface of the substrate, and then a functional group such as a mercapto group is present at both ends of the PEG. PEG is supported. Therefore, since the functional groups at both ends are bonded to the base material to form a looped PEG, there is a disadvantage that the target surface with suppressed nonspecific adsorption is less likely to be obtained. Further, these conventional techniques cannot suppress nonspecific adsorption of biomolecules having a small molecular weight.
Therefore, adsorption of low-molecular-weight biomolecules, which could not be suppressed even with high molecular weight PEG, can be suppressed, and a minimal region can be created by a simple method without the trouble of modifying two types of polymers through steps. Development of molecular layers that can be modified is desired.

  Under such circumstances, the present invention suppresses nonspecific adsorption of biomolecules having various molecular weights from high to low molecules as much as possible, and can detect the target molecular species with high sensitivity. It is an object of the present invention to provide a adsorptive adsorption suppressing material.

As a result of diligent research, the present inventors have found that the above-mentioned problem is caused by forming a monomolecular film comprising a PEG derivative having an alkylene group having a specific carbon number in the molecule and an ethylene glycol repeating unit on the surface of the substrate. The present invention has been found out to be solved, and the present invention has been completed.
That is, in the present invention, the following configurations 1 to 6 are adopted.
1. A non-specific adsorption suppressing material formed by forming a monomolecular film of a compound represented by the general formula (1) on the surface of a substrate.
_{HO (CH 2 CH 2 O)} m - (CH 2) n -SH (1)
In the formula, m represents an integer of 1 to 3, and n represents an integer of 2 to 8.
2. 2. The nonspecific adsorption-suppressing material according to 1, wherein the substrate is composed of a material selected from conductive metals and metal oxides.
3. The non-specificity according to 1 or 2, wherein the monomolecular film is a mixed monomolecular film of a compound represented by the general formula (1) and a molecule that recognizes and binds to a target molecule. Adsorption suppression material.
4). 4. The nonspecific adsorption-suppressing material according to 3, wherein the molecule that recognizes and binds to the target molecule is a molecule having a thiol group (—SH) at the end of the molecule.
5. The nonspecific adsorption-suppressing material according to 3 or 4, wherein the molecule that recognizes and binds to the target molecule is selected from sugars, aptamers, and nucleic acids.
6). 6. The nonspecific adsorption suppressing material according to any one of 3 to 5, wherein the ratio of molecules that recognize and bind to the target in the mixed monolayer is 0.1 to 60%.

The present invention provides non-specific adsorption inhibition comprising a highly flexible and hydrophilic ethylene glycol repeating unit in one molecule and a hydrophobic alkyl chain portion having a specific length for increasing the molecular density. Since a molecule-modified monomolecular layer can be constructed, a highly dense molecular layer can be easily produced. As a result, it is possible to easily suppress non-specific adsorption of low-molecular biomolecules that could not be suppressed by modification with high molecular weight PEG. In addition, it is possible to form a modified layer that suppresses nonspecific adsorption of biomolecules on the surface of a very small substrate that was difficult to form with conventional polymer PEG derivatives.
Furthermore, there is a problem in detecting the target protein, nucleic acid, peptide, etc. by forming a mixed monolayer of the compound represented by the general formula (1) and a molecule that recognizes and binds to the target molecule. Since it becomes possible to detect only target molecules and substances with high sensitivity by lowering the noise level due to non-specific adsorption, it will be an important technology for making various chips such as protein chips and DNA chips. is there.

As the base material constituting the nonspecific adsorption suppressing material of the present invention, gold, platinum, silver, other metal electrodes other than copper, semiconductor electrode base materials such as silicon, and transparent conductive electrodes such as indium tin oxide (ITO) Are advantageously used, but it is particularly preferred to use conductive metals and metal oxides.
These metals, semiconductors, and the like are known that organic compounds containing sulfur form metal-S bonds in a self-organizing manner and stably form a modified film. The shape and size of the base material are arbitrary, and various sizes of plate-like base materials and particulate base materials can be used.

In the present invention, a monomolecular film of a nonspecific adsorption-suppressing molecule comprising a compound represented by the following general formula (1) is formed on the surface of these base materials.
_{HO (CH 2 CH 2 O)} m - (CH 2) n -SH (1)
In the formula, m represents an integer of 1 to 3, and n represents an integer of 2 to 8.
As the compound represented by the general formula (1), a dimerized compound in which the -SH group of the compound is oxidized to a disulfide group (-SS-) may be used. These compounds can be produced, for example, according to the production examples described later.

In order to construct a modified monolayer with the above functional molecules on the surface of a solid substrate, nonspecific adsorption-suppressing molecules are dispersed in a solution (aqueous solution or water-ethanol mixed solution) in advance and the substrate is modified thereby. . The molecular layer is spontaneously self-assembled and arranged on the substrate surface. The modified layer immobilized on the substrate can almost completely suppress non-specific adsorption of various biomolecules ranging from several hundred thousand to several hundreds of proteins to peptides.
This non-specific adsorption-suppressing molecule is based on (a) a mixture solution together with a molecule that recognizes and binds to a target molecule (alkanethiol, aptamer, single-stranded nucleic acid, etc. having a sugar chain). A mixed monomolecular film is constructed on the substrate surface by modifying the surface of the material, or (b) using a two-stage method in which the target recognition molecule is adsorbed in advance and then reacted with a non-specific adsorption inhibiting molecule solution. be able to. When this mixed monolayer is used, the noise level response due to nonspecific adsorption of biomolecules other than the target molecule is suppressed, and the target molecule (for example, lectin for sugar, protein for aptamer, single protein) In the case of a strand nucleic acid, the response of a nucleic acid having a complementary base pair sequence can be detected with high sensitivity.

As a target recognition molecule constituting this mixed monomolecular film, a molecule having a thiol group (-SH) at at least one end of the molecule, or a disulfide group (-SS-) is formed by oxidizing the thiol group. It is preferred to use a molecule that is quantified.
Examples of such target recognition molecules include 12-mercaptododecyl-β-maltoside (a maltoside group specifically recognizes Con A), a sugar having a mercapto group or a disulfide group at its terminal, and various proteins. Examples include aptamers having the above functional group at the terminal to be recognized, and nucleic acids having the above functional group at the terminal having a complementary base pair sequence to those that recognize a single-stranded nucleic acid.
The non-specific adsorption-suppressing molecule constituting the mixed monolayer is not only a compound in which _n in — (CH ₂ ) _n — is an integer of 2 to 8 in the above general formula (1), A compound that is an integer of 11 can also be used.

  In the mixed monomolecular film, the molecular species capable of molecular recognition is preferably about 0.1 to 60% of the molecular species constituting the film. For example, when a sugar chain-lectin is used as a target molecule, 0.1 to 60%, aptamer is set to 0.1 to 57%, and single-stranded nucleic acid is set to about 0.1 to 50%. It becomes possible to detect the response with reduced efficiency. For example, when a binding (adsorption) reaction due to interaction occurs, the response detection can be performed by tracking a change in the refractive index of the surface with a surface plasmon resonance sensor (SPR sensor). Since non-specific adsorption does not occur in a membrane that does not include a recognition site (a membrane containing only non-specific adsorption-suppressing molecules), almost no SPR response is detected.

EXAMPLES Next, although an Example demonstrates this invention further in detail, the following specific examples do not limit this invention.
In the following example, a compound represented by the formula (HO (CH ₂ CH ₂ O) ₃ — (CH ₂ ) ₂ —SH) is converted to PEGC2SH, (HO (CH ₂ CH ₂ O) ₃ — (CH ₂ ) ₄ — The compound represented by the formula (HO (CH ₂ CH ₂ O) ₃ — (CH ₂ ) _n —SH) is represented by the number of carbon atoms of — (CH ₂ ) _n — as in PEGC4SH. Is abbreviated as PEGCnSH.
The properties of the monomolecular film formed on the substrate surface were evaluated by measuring changes in the refractive index of the surface with a surface plasmon resonance sensor (Biacore T100 system or Biacore 2000 system).

(Production Example 1)
(Synthesis of non-specific adsorption inhibiting molecule triethylene glycol alkyl thiol PEGCnSH)
(1) tetraethylene glycol monomethyl thiol [triethylene glycol C2 thiol _{(HO (CH 2 CH 2 O} ) 3 - (CH 2) 2 -SH, PEGC2SH)] Synthesis (1-1) of tetraethylene glycol mono tosylate Composition

Tetraethylene glycol 9.70 g (50 mmol), triethylamine 2.02 g (20 mmol), and tetrahydrofuran (THF) 150 mL were placed in a three-necked flask (300 mL), and the mixture was stirred at 0 ° C. 20 mL of a THF solution of 1.91 g (10 mmol) of p-toluenesulfonyl chloride (TsCl) was added dropwise, and the mixture was further stirred at room temperature for 4 days. To the reaction solution, 300 mL of 5 wt% hydrochloric acid was poured and extracted with 200 mL of chloroform. Chloroform and THF were distilled off, and the target product was purified by gel permeation chromatography (GPC). A colorless liquid (C ₁₅ H ₂₄ O ₇ S, molecular weight M = 348.37) was obtained with a yield of 82%.

(1-2) Synthesis of monothioacetyltetraethylene glycol

In a three-necked flask (300 mL), 3.48 g (10 mmol) of tetraethylene glycol monotosylate obtained in (1-1) above and 100 mL of DMF were placed in a nitrogen atmosphere and stirred at room temperature. 3.43 g (30 mmol) of potassium thioacetate was added, and the mixture was further stirred at room temperature for 12 hours. To the reaction solution, 200 mL of 5 wt% hydrochloric acid was poured and extracted with 100 mL of chloroform. Chloroform and dimethylformamide (DMF) were distilled off, dried under reduced pressure, and used directly in the next reaction. Colorless liquid _{(C 10 H 20 O 5 S} , M = 252.33) was obtained in 98% crude yield.

(1-3) triethylene glycol C2 thiol _{(HO (CH 2 CH 2 O} ) 3 - (CH 2) 2 -SH, PEGC2SH) Synthesis of

In a three-necked flask (300 mL), 2.52 g (10 mmol) of monothioacetyltetraethylene glycol obtained in (1-2) above and 200 mL of ethanol were placed in a nitrogen atmosphere and stirred at room temperature. 1.12 g (10 mmol) of t-butyl potassium was added, and the mixture was further stirred at room temperature for 3 hours. 400 mL of 5 wt% hydrochloric acid was poured into the reaction solution, and extracted twice with 200 mL of chloroform. Chloroform and ethanol were distilled off, and the target product was purified with a silica gel column (developing solvent, chloroform: methanol = 100: 1). A colorless liquid (C ₈ H ₁₈ O ₄ S, M = 210.29) was obtained with a yield of 59%.

[Production Example 2]
(2) triethylene glycol C4 thiol _{(HO (CH 2 CH 2 O} ) 3 - (CH 2) 4 -SH, PEGC4SH) Synthesis of (2-1) 4-bromobutyl triethylene glycol

In a three-necked flask (300 mL), 2.16 g (10 mmol) of 1,4-dibromobutane, 15.0 g (100 mmol) of triethylene glycol and 200 mL of THF were added and stirred at room temperature. 1.12 g (10 mol) of t-butyl potassium was added, and the mixture was heated to reflux for 6 hours. After standing to cool, THF was distilled off, 200 mL of 5 wt% hydrochloric acid was poured, and the mixture was extracted with 100 mL of chloroform. The chloroform layer was washed with 200 mL of water. Chloroform was distilled off and purified by a silica gel column (developing solvent, chloroform: methanol = 100: 1). Colorless liquid _{(C 10 H 21 O 4 Br} , M = 285.18) was obtained in 28% yield.

(2-2) Synthesis of 4-bromobutyltriethylene glycol benzoyl ester

In a three-necked flask (300 mL), 2.85 g (10 mmol) of 4-bromobutyltriethylene glycol obtained in (2-1) above, 2.52 g (25 mmol) of triethylamine, and 100 mL of THF were added, and the mixture was stirred at 0 ° C. 20 mL of a THF solution of 2.81 g (20 mmol) of benzoyl chloride was added dropwise, and the mixture was further stirred at room temperature for 24 hours. To the reaction solution, 200 mL of 5 wt% hydrochloric acid was poured and extracted with 100 mL of chloroform. Chloroform and THF were distilled off, and the target product was purified by GPC. Colorless liquid _{(C 17 H 25 O 5 Br} , M = 389.29) was obtained in 87% yield.

(2-3) Synthesis of 4-thioacetylbutyl triethylene glycol benzoyl ester

In a three-necked flask (100 mL), 779 mg (2 mmol) of 4-bromobutyltriethylene glycol benzoyl ester obtained in (2-2) above and 50 mL of DMF were added under a nitrogen atmosphere, and the mixture was stirred at room temperature. 684 mg (6 mmol) of potassium thioacetate was added, and the mixture was further stirred at room temperature for 4 hours. To the reaction solution, 100 mL of 5 wt% hydrochloric acid was poured and extracted with 50 mL of chloroform. Chloroform and DMF were distilled off, and the target product was purified by GPC. A colorless liquid (C ₁₉ H ₂₈ O ₆ S, M = 384.49) was obtained with a yield of 72%.

(2-4) triethylene glycol C4 thiol _{(HO (CH 2 CH 2 O} ) 3 - (CH 2) 4 -SH, PEGC4SH) Synthesis of

In a three-necked flask (100 mL), under a nitrogen atmosphere, 769 mg (2 mmol) of 4-thioacetylbutyltriethylene glycol obtained in (2-3) above and 80 mL of ethanol were added and stirred at room temperature. 224 mg (2 mmol) of t-butyl potassium was added, and the mixture was further stirred at room temperature for 3 hours. To the reaction solution, 200 mL of 5 wt% hydrochloric acid was poured and extracted with 100 mL of chloroform. Chloroform and ethanol were distilled off, and the target product was purified with a silica gel column (developing solvent, chloroform: methanol = 100: 0 to 1). Colorless liquid _{(C 10 H 22 O 4 S} , M = 238.34) was obtained in 72% yield.

[Production Examples 3 to 5]
(3) Triethylene glycol C6~C11 thiol _{(HO (CH 2 CH 2 O} ) 3 - (CH 2) n -SH, PEGC8SH: n = 6~11) of Synthesis of (2-1) to (2-4 ), Triethylene glycol C6 thiol (HO (CH ₂ CH ₂ O) _3- (CH ₂ ) ₆ -SH, PEGC6SH), triethylene glycol C8 thiol (HO (CH ₂ CH ₂ O ) _3- (CH ₂ ) ₈ -SH, PEGC8SH) and triethylene glycol C11 thiol (HO (CH ₂ CH ₂ O) _3- (CH ₂ ) ₁₁ -SH, PEGC11SH) were synthesized.

[Example 1]
(Relationship between inhibitory intramolecular alkyl chain length and protein adsorption inhibition ability)
Different non-specific adsorption inhibition molecular alkyl chain length obtained in each Production Example _{(PEGCnSH: HO (CH 2 CH} 2 O) 3 - (CH 2) n -SH, n = 2, 4, 6, 8, 11) 1 μM aqueous solution or water-ethanol mixture (ethanol 20%) was prepared, and flowed for 2500 seconds at a flow rate of 10 μL / min, and immobilized on a gold substrate to form a monomolecular film. The thiol group introduced into the non-specific adsorption-suppressing molecule contacts the gold substrate and simultaneously forms a gold-SH bond and is adsorbed on the gold substrate surface. The amount of nonspecific adsorption-suppressing molecules measured by Biacore T-100 was 613RU (n = 2), 622RU (n = 4), 866RU (n = 6), 1392RU (n = 8), 905RU (n = 11) (RU: Resonance unit, 1000RU = 1 ng / mm ² ).

Concanavalin A (Con A), a lectin solution (1 μM, HEPES buffer (pH 7.4)) was allowed to flow for 2000 seconds at a flow rate of 10 μL / min on the surface after each nonspecific adsorption-suppressing molecule was adsorbed. The adsorption amount was measured by switching to a buffer solution not contained, and the results are shown in FIG. For comparison, FIG. 1 also shows the results of measurement of a carboxymethyl dextran coating layer chip (CM5) that is usually used as a standard.
As can be seen in FIG. 1, a change (adsorption) of 29.1 RU was confirmed when n = 2, compared with the adsorption (125.8 RU) observed for the chip (CM5) with a carboxymethyl dextran coating layer. It can be seen that the adsorption is suppressed to 1 / min or less. The effect of suppressing non-specific adsorption of Con A becomes more prominent as the alkyl chain length is longer, and on the surface of the non-specific adsorption-suppressing molecular layer when n = 11, the adsorption of Con A is 1.7 RU, which is completely adsorbed. It was confirmed that it was suppressed.

[Example 2]
(Electrochemical evaluation of the density of each nonspecific adsorption inhibiting molecule)
As the alkyl chain length becomes longer, the density of molecules in the nonspecific adsorption-suppressing molecular film increases, and it is considered that the effect of suppressing nonspecific adsorption of proteins is further enhanced.
Therefore, the density of the molecular layer by each non-specific adsorption inhibiting molecule was electrochemically evaluated as follows.
A commercially available gold single crystal electrode is immersed in a solution of each molecule of PEGCnSH (n = 2, 4, 6, 8, 11) (1mM water-ethanol mixed solution) for 1 hour, and non-specific adsorption-suppressing molecules on the electrode surface A monomolecular film was prepared. This modified layer is swept in 0.5 M potassium hydroxide aqueous solution from -0.5 V to -1.2 V to reductively desorb molecules from the gold surface. The results are shown in FIG.

This reaction is represented by the following formula, and one electron is involved in the reductive elimination of one molecule. (Reference: Y. Sato et.al, Electroanalysis, 17, 965 (2005); MM Walczak, et. Al., Langmuir, 7, 2687 (1991), etc.)
^{RS-Au + e - → RS} - + Au
As can be seen from FIG. 2, the amount of electricity increases as the alkyl chain length increases. That is, as the alkyl chain length increases, the molecular density per unit area increases, and it can be said that this effect further enhances the protein suppression effect.

Example 3
(Con A recognition by non-specific adsorption-inhibiting molecules and mixed adsorption layer containing maltoside group, improvement of signal-noise level)
12-mercaptododecyl-β-maltoside (the maltoside group specifically recognizes Con A) and non-specific adsorption-inhibiting molecules of various alkyl chain lengths [PEGCnSH (n = 2, 4, 6, 8, 11)] Using a mixed solution [12-mercaptododecyl-β-maltoside: PEGCnSH (n = 2, 4, 6, 8, 11) = 1: 9 (molar ratio), 0.1 mM water-ethanol mixture (ethanol 20%)] Then, a monomolecular film in which 12-mercaptododecyl-β-maltoside and nonspecific adsorption-inhibiting molecules of various alkyl chain lengths were co-adsorbed on a gold substrate was formed in the same procedure as in Example 1. FIG. 3 shows the results of measuring the recognition effect (adsorption amount) of Con A in this monomolecular film using the Biacore 2000 system.
Fig. 3 (a) shows the results of examining the recognition effect of Con A on this membrane (Con A adsorption amount). The light-colored graph on the left side of each nonspecific adsorption-suppressing molecule is a modified membrane containing only PEGCnSH molecules (PEGCnSH). (N = 2, 4, 6, 8, 11) Shows non-specific adsorption amount of Con A in 0.1 mM water-ethanol mixture (ethanol 20%). Represents the amount of Con A adsorbed on a monomolecular film. According to this figure, it can be seen that the amount of adsorption is almost equal from n = 2 to 6, and Con A is well recognized even when n = 8.

FIG.3 (b) represents the signal-noise ratio (S / N ratio) in the mixed monomolecular film of each nonspecific adsorption | suction suppression molecule | numerator of Fig.3 (a).
As can be seen in FIG. 3B, nonspecific adsorption is suppressed as the alkyl chain length of the functional molecule co-adsorbed with the maltoside molecule increases, and as a result, a significant improvement in the signal-to-noise ratio can be realized. . In the case of n = 11, the length of the non-specific adsorption-suppressing molecule greatly exceeds the height of the maltoside group that is the molecular recognition site [see FIG. 4 (C)]. Since the response is also greatly reduced, the signal-to-noise ratio is reduced (see FIG. 3 (a)). On the other hand, when the nonspecific adsorption-suppressing molecule of n = 2 to 8 of the present invention is used, the signal-noise ratio is large and the target molecule can be detected with high sensitivity.
A schematic diagram of the mixed monomolecular film at this time is shown in FIG. 4A shows a mixed monomolecular film using PEGC2SH and 12-mercaptododecyl-β-maltoside, FIG. 4B shows a mixed monomolecular film using PEGC6SH and 12-mercaptododecyl-β-maltoside, and FIG. 4 (C) is a schematic diagram of a mixed monolayer using PEGC11SH and 12-mercaptododecyl-β-maltoside. As shown in FIG. 4C, in the mixed monomolecular film using PFGC11SH, the length of the nonspecific adsorption-suppressing molecule greatly exceeds the height of the maltoside group that is the molecular recognition site. The signal-to-noise ratio is small because the signal response is greatly reduced even if the value is lowered.

Example 4
(Adsorption suppression effect of biomolecules with various molecular weights)
Using the non-specific adsorption suppressing material (n = 2, 4, 6, 8, 11) of the present invention obtained in Example 1, non-specific adsorption of biomolecules of various molecular weights in each suppressing material The amount was measured using a Biacore T-100 system and the results are shown in Table 1.
For comparison, the surface of a gold substrate modified with a commercially available high molecular weight polyethylene glycol (molecular weight 2000) or a commercially available product A (Nanobiotec Co., Ltd., a metal surface blocking agent), a chip (CM5) coated with carboxymethyldextran, and The results measured on the modified gold surface are also listed.

As shown in Table 1, Con A with a molecular weight of 104,000, bovine serum albumin (BSA) with a molecular weight of 66,300, 17mer [base sequence: ATT TCT TGC TTA AAG TC (SEQ ID NO: 1), molecular weight 5100.43]], 36mer [base sequence] : TGC CCT GGA CCT GCG AAA TCC AGA ACA AAG CAC (SEQ ID NO: 2), molecular weight 11032.29] It was confirmed that nonspecific adsorption of biomolecules such as nucleic acids was suppressed to a very low value. In addition, various peptides such as angiotensin (molecular weight 1296.5), bradykinin (molecular weight 1060.2), and further low molecular weight cell adhesion oligopeptides: arginine-glycine-aspartic acid-serine (RGDS) (molecular weight 433.42), Ac-Asp It was confirmed that non-specific adsorption was suppressed almost completely up to -Glu (molecular weight 304.25), and the effectiveness of the non-specific adsorption-suppressing material was verified.
Compared with a surface modified with a commercially available high molecular weight polyethylene glycol (molecular weight 2000) or a commercially available product A (metal surface blocking agent Nanobiotech Co., Ltd.), this effect is from a high molecular weight to a low molecular weight, particularly several hundreds of molecular weights. It was confirmed that it was effective in suppressing non-specific adsorption of low molecular weight biomolecules.

Example 6
(Recognition of thrombin by mixed adsorption layer of non-specific adsorption suppression molecule and thrombin recognition nucleic acid aptamer molecule, improvement of signal-noise level)
Nucleic acid aptamer that recognizes thrombin [HS-C6-TTT TTT TTT TTT GGT TGG TGT GGT TGG (SEQ ID NO: 3), molecular weight 8814.49] and nonspecific adsorption-suppressing molecule similar to Example 1 (n = 2 to 11) Was used to form an aptamer mixed monomolecular film on the surface of a Biacore gold chip by the following procedure. C6 of the nucleic acid aptamer means (CH ₂ ) ₆ . FIG. 5 shows the results of measuring the amount of thrombin adsorbed on the obtained co-adsorbed layer using the Biacore system T-100. Further, FIG. 6 shows the result of comparing the signal noise level.
Use HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% Tween 20) / 50 mM KCL solution (pH 7.4) as the solvent, and adjust the nucleic acid aptamer to 250 nM according to the amount of adsorption. Flowed for 5 minutes each time, the initial amount of aptamer adsorption was controlled. Thereafter, a 0.1 mM solution of each PEGCnSH molecule was flowed at a flow rate of 5 μl / min for 1 hour to construct an aptamer mixed monolayer. For comparison, an example in which mercaptoethanol (ME), which is a hydroxyl-terminated thiol commonly used in experiments, is used as a nonspecific adsorption-suppressing molecule is also shown.

In each modified monolayer, the amount of aptamer immobilized should be approximately constant at 2.1-2.7 × 10 ¹² molecules / cm ^2, and nonspecifically adsorbed molecules at 1.0-1.5 × 10 ¹⁴ molecules / cm ² A mixed monolayer was prepared as described above. The left side of FIG. 5 shows the amount of nonspecific adsorption (noise) occurring on the surface of each nonspecifically adsorbed molecule alone.
Even when ME is used, the nonspecific adsorption of thrombin is suppressed to a relatively low level as shown in the figure, but the nonspecific adsorption suppression molecule (n = 2-8) of the present invention has a nonspecific adsorption. It has been confirmed that the inhibitory effect is further suppressed. When PEGC2SH was used as a non-specific adsorption molecule, it was confirmed that thrombin adsorption was much better. In the case of hydroxyl terminal (ME), the SN ratio was about 5, whereas when each PEGCnSH was used, it was 50 times or more, and when PEGC6SH was used, it was 147. It can be seen that PEGC6SH is 26 times better than the hydroxyl-terminated alkanethiol molecule. (See Figure 6)

It is a figure which shows the adsorption amount of concanavalin A to a non-specific adsorption | suction suppression molecule | numerator PEGCnSH (n = 2, 4, 6, 8, 11) modification layer and a commercially available CM5 chip | tip (Biacore system, GE Healthcare Science company). The vertical axis represents the RU value (RU: resonance unit, 1000 RU = 1 ng / mm ² ) indicating the amount of adsorption. Measured with Biacore T-100 system. Nonspecific adsorption-inhibiting molecule PEGCnSH (n = 2, 4, 6, 8, 11) The amount of adsorption of each molecule determined from the electric quantity data accompanying reductive desorption from the gold surface of the modified layer, It is a figure which shows the relationship of alkyl chain length (n). (A) It is a figure which shows the adsorption amount of Con A in the molecular film which co-adsorbed 12-mercaptododecyl-β-maltoside (the maltoside group specifically recognizes Con A) and PEGCnSH molecules of various alkyl chain lengths. Measured with Biacore 2000 system. The open graph shows the non-specific adsorption result of Con A on the modified membrane of only the PEGCnSH functional molecule. (B) It is a figure which shows the ratio of the adsorption response (signal) of Con A on a maltoside coexistence molecule recognition film | membrane, and the nonspecific adsorption response (noise) in each PEGCn molecular layer. It is a schematic diagram of a molecular film in which 12-mercaptododecyl β-maltoside and PEGCnSH functional molecules having various alkyl chain lengths are co-adsorbed. (A) 12-mercaptododecyl β-maltoside and PEGC ₂ SH mixed monolayer, (B) 12-mercaptododecyl β-maltoside and PEGC ₆ SH mixed monolayer, (C) 12-mercaptododecyl A schematic diagram of a mixed monolayer of β-maltoside and PEGC ₁₁ SH is shown. The figure shows the amount of thrombin adsorbed on a coadsorption layer modified with a nucleic acid aptamer that recognizes thrombin and the nonspecific adsorption inhibitor molecule (PEGCnSH) and a coadsorption layer of mercaptoethanol (ME) and a nucleic acid aptamer. is there. Measurement is Biacore T-100 system. The vertical axis indicates the amount of thrombin adsorption (RU value). The left (black) data in the graph is the amount of thrombin adsorbed on the surface of nonspecific adsorption-suppressing molecules only. The right (gray) shows the adsorption response of thrombin in aptamer and each non-specific adsorption-inhibiting molecule mixed monolayer. It is a figure which shows the ratio of the adsorption amount (signal) of the thrombin in the aptamer film | membrane shown in FIG. 5, and the nonspecific adsorption response (noise) of the thrombin in the film | membrane (PEGCnSH, ME) which does not contain an aptamer.

Claims (6)

A non-specific adsorption suppressing material formed by forming a monomolecular film of a compound represented by the general formula (1) on the surface of a substrate.
_{HO (CH 2 CH 2 O)} m - (CH 2) n -SH (1)
In the formula, m represents an integer of 1 to 3, and n represents an integer of 2 to 8.   The non-specific adsorption suppressing material according to claim 1, wherein the base material is composed of a material selected from conductive metals and metal oxides.   The non-molecular film according to claim 1 or 2, wherein the monomolecular film is a mixed monomolecular film of a compound represented by the general formula (1) and a molecule that recognizes and binds to a target molecule. Specific adsorption suppression material.   The nonspecific adsorption-suppressing material according to claim 3, wherein the molecule that recognizes and binds to the target molecule is a molecule having a thiol group (-SH) at the end of the molecule.   The nonspecific adsorption-suppressing material according to claim 3 or 4, wherein the molecule that recognizes and binds to the target molecule is selected from sugars, aptamers, and nucleic acids.   6. The nonspecific adsorption-suppressing material according to claim 3, wherein a ratio of molecules that recognize and bind to the target in the mixed monolayer is 0.1 to 60%. .